de Iongh Lab ~ Current Research Projects

• Expression and role of integrin signalling via FAK and ILK during lens development and EMT of cataract

• Expression and role of receptors for the TGF-ß superfamily during lens development

• The role of the Wnt/ß-catenin pathway in lens development

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Project 1 - Integrins in lens development

The lens expresses mainly laminin-binding integrins during normal development and growth. Expression of these integrins is regulated by growth factors, such as FGF, in the ocular media. Studies of knockouts indicate that loss of individual integrins has little or no effect on lens development as they appear to act redundantly. However, double mutations can result in disruption of the lens, suggesting they act cooperatively.

alpha3 integrin localisation in lens whole-mounts shows it is restricted to epithelial and is rapidly "turned off" in differentiating fiber cells that start to express ß-crystallin near the equator of the lens.

Double mutants of alpha3 and alpha6 integrin show disrupted epithelial layer and lens capsule at E13. By embryonic day 14, the lens appears to be completely absent. Tissues courtesy of Dr Elizabeth Labouesse.

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FAK & ILK in Lens Development

Expression of FAK and ILK in the lens (purple) by in situ hybridisation.

Focal adhesion kinase (FAK) and integrin-linked kinase (ILK) are key molecules involved in the transduction of signals by integrins from the extracellular matrix. Both kinases are expressed in the eye during development and are regulated by growth factors. FGF regulates the expression of FAK during development and TGF-ß regulates the expression of ILK during cataract formation.

Our current studies aim to investigate the functional roles of these molecules in development and cataract by manipulating the expression and activation of these kinases in cultures of lens epithelial cells and also in transgenic mice.

Initial studies suggest that over-activation of ILK may be involved in cataract formation (epithelial-mesenchymal transition).

Transfection of lens cells with an active form of ILK (D-F) causes the cells to undergo an EMT similar to that seen in cataract. Control transfected cells remain as cuboidal epithelial cells (A-C).

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Project 2 - TGF-ß superfamily Receptors in Lens Development

We have shown that the lens expresses activin, BMP and TGF-ß receptors. Inhibition of TGF-ß receptor signalling by ectopic expression of dominant-negative receptors in transgenic mice results in disruption of terminal fiber differentiation. Analysis of these lenses shows that there is aberrant expression of BMP receptors possibly as compensatory response to the loss of TGF-ß signalling.

Current studies are focussing on the roles of the TGF-ß superfamily in lens development.

In transgenic mice that express mutant TGFß receptors in the lens, there appears to be a compensatory up-regulation of BMP type I receptor (BMPRIA/Alk3) both at mRNA and protein level.

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Project 3 - Wnt/ß-catenin pathway in lens development

Recent studies implicate the Wnt/ß-catenin pathway in the development of the lens and retina. Current studies in the lab show that conditional mutations of ß-catenin and APC genes in the lens results in disruption of the lens structure and expression of epithelial genes. Further studies on these conditional mutants are underway.

Conditional mutation of the ß-catenin gene in the lens results in disrupted lens development.